Identification of easily detectable biomarkers for TB is a global health priority. With an estimated 8.8 million new cases of active tuberculosis (TB) occurring in 2010 TB remains a global public health problem. Rapid TB diagnosis is critical for early treatment initiation and reduction of transmission, morbidity, and mortality. Current rapid diagnostic tests lack either sensitivity (e.g. sputum microscopy) or cannot be easily applied in resource-limited settings (e.g., nucleic acid detection) where the vast majority of TB cases occur. Serum antibodies (Abs) can be rapidly detected by methods that can be turned into simple dip-stick formats. TB serology, however, has suffered from decades of unsuccessful attempts to develop accurate tests for TB. We have recently demonstrated that pathogenic mycobacteria produce membrane vesicles (MVs) that contribute to mycobacterial virulence in mice. These vesicles provide an effective way to release immune-modulatory factors and the host immune responses to MVs provide a unique opportunity for identification of novel biomarkers. Our preliminary data indicate that 3 proteins enriched in MVs of pathogenic mycobacteria induce a highly sensitive and specific Ab biomarker signature for TB. Therefore, our overarching hypothesis is that the simultaneous Ab response to specific MV proteins is strongly and significantly associated with TB. Consequently, we propose to use our well-characterized serum sample collection to develop these biomarkers for use in a rapid point-of-care (POC) test applicable in resource-limited settings. Our specific aims are: 1) To characterize Ab responses to mycobacterial MVs in patients suspected of having TB; and 2) To identify the mycobacterial MV proteins that elicit a TB Ab biomarker signature, express them in vitro, and verify Ab responses for diagnostic assay development. This is a highly feasible project with robust preliminary data and an innovative approach to address the need for a simple, rapid TB diagnostic. The ultimate goal of this proposal is the development of an assay that could lead to a POC test with the potential of replacing sputum microscopy for the diagnosis of TB.